1. Field of the Invention
This invention concerns an LED excitation source and module useful for full plate imaging fluorescence instruments as well as methods for calibrating such instruments.
2. Description of Related Art
Spectroscopy involves the study of matter using electromagnetic radiation. Spectroscopic measurements can be separated into three broad categories: absorbance, scattering/reflectance, and emission. Absorbance assays involve relating to the amount of incident light that is absorbed by a sample to the type and number of molecules in the sample. Absorbance assays are a powerful method for determining the presence and concentration of an analyte in a sample. Most commonly, absorbance is measured indirectly by studying the portion of incident light that emerges from the sample. Scattering assays are similar to absorbance in that the measurement is based on the amount of incident light that emerges or is transmitted from the sample. However, in the case of scattering, the signal increases with the number of interactions, whereas, in the case of absorbance, the signal is inversely proportional to the interactions. Emission assays look at electromagnetic emissions from a sample other than the incident light. In each case, the measurements may be broad spectrum or frequency specific depending on the particular assay. Most commonly, emission assays involve the measurement of luminescence. The techniques of absorbance, scattering/reflectance, and luminescence are described in detail in the following patent applications, which are hereby incorporated by reference in their entirety for all purposes: WIPO Publication No. WO 00/06991, published Feb. 10, 2000; and corresponding U.S. patent application Ser. No. 09/765,869, filed Jan. 19, 2001.
Luminescence is a preferred assay technique due to its specificity and sensitivity, among others. Luminescence is the emission of light from excited electronic states of atoms or molecules. Luminescence generally refers to all kinds of light emission, except incandescence, and may include photoluminescence, chemiluminescence, and electrochemiluminescence, among others. In photoluminescence, which includes fluorescence and phosphorescence, the excited electronic state is created by the absorption of electromagnetic radiation. In chemiluminescence, which includes bioluminescence, the excited electronic state is created by a transfer of chemical energy. In electrochemiluminescence, the excited electronic state is created by an electrochemical process.
Luminescence assays are assays that use luminescence emissions from luminescent analytes to study the properties and environment of the analyte, as well as binding reactions and enzymatic activities involving the analyte, among others. In these assays, the analyte itself may be the focus of the assay, or the analyte may simply act as a reporter that provides information about another material or target substance that is the true focus of the assay. Recently, luminescence assays have been used in high throughput procedures to screen pharmaceutical drug candidate libraries for drug activity and to identify single-nucleotide polymorphisms (SNPs).
Luminescence assays may involve detection and interpretation of one or more properties of the luminescence or associated luminescence process. These properties may include intensity, excitation and/or emission spectrum, polarization, lifetime, and energy transfer, among others. These properties also may include time-independent (steady-state) and/or time-dependent (time-resolved) properties of the luminescence. Representative luminescence assays include fluorescence intensity (FLINT), fluorescence polarization (FP), fluorescence resonance energy transfer (FRET), fluorescence lifetime (FLT), total internal reflection fluorescence (TIRF), fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), and bioluminescence resonance energy transfer (BRET), among others.
Luminescence assays have been conducted using various light sources, including arc lamps and lasers. Unfortunately, these light sources suffer from a number of shortcomings. The gas used in arc lamps typically is under high pressure, so that explosion is always a danger, and the associated power supplies may produce transients that can damage other electronic components of the system. The lifetime of arc lamps may be short, so that the lamps must be changed frequently. Moreover, typical arc lamps (including flash arc lamps) suffer from intensity instability, with short term noise of several percent, which is much worse than good lasers such as laser diodes and diode pumped solid-state lasers, with short-term noise of typically less than 0.5%. In addition, lamps have a slow and steady long-term decay of intensity, whereas lasers normally exhibit a constant intensity up until catastrophic failure. The spectral output of some arc lamps and most lasers is very limited, so that desired excitation wavelengths may not be available. For example, two commonly used light sources, the mercury arc lamp and the argon-ion laser, produce significant visible light only at two wavelengths below about 550 nm. Moreover, the procedure for switching or tuning between these wavelengths can be so cumbersome and impractical that some experimentalists have resorted to the expensive alternative of incorporating multiple lasers into their instrumental setups. Significantly, an ability to use and switch between various excitation wavelengths would permit use of a wider variety of dyes, which in turn would facilitate the development of new luminescence assays, including new high throughput cell-based luminescence assays.
Luminescence assays also have been conducted using various detection schemes. These schemes may require alignment of a sample and portions of an optical relay structure (such as an optics head) for directing light to and from the sample. This alignment typically is accomplished by physically moving the sample relative to the optical relay structure and/or by physically moving the optical relay structure relative to the sample. This movement may be followed by a waiting period before measurement to allow vibrations to subside. Time spent during alignment and subsequent waiting periods is downtime because it is time during which data cannot be collected from the sample. This downtime is especially significant in high-throughput screening, where tens or hundreds of thousands of samples must be aligned with an optical relay structure to conduct a particular study.
In principle, reading simultaneously from a plurality of samples or from a larger area of a single sample can reduce the number of alignment steps and thus the amount of downtime in these assays. Indeed, instrumentation has been developed that directs light from an arc lamp or from a continuous wave laser tuned to a single fixed wave length to multiple wells of a microplate using a mechanical (e.g., rotating polygon or galvanometric) scanner and/or a wide-field illuminator. However, reading with the scanner is slow, because samples are analyzed well by well, and reading with the widefield imager reduces intensities, because excitation light is distributed to areas between or outside the samples. Reduced intensities may decrease signal-to-noise ratios, decreasing reliability, especially with less intense nonlaser light sources. Prior art instrument systems also may be limited because it may be difficult to change the emission filter to correspond to a change in excitation wavelength. This is especially true with simultaneous reading because filters for simultaneous reading may need to be quite large to filter emission light passing from large-area samples, such as microplates, to large-area imaging devices, such as charge-coupled devices (CCDs), charge injection device (CID) arrays, videcon tubes, photomultiplier tube arrays, position sensitive photomultiplier tubes, and the like. Significantly, it is desirable to increase the number of measurements made in a given time period. Increased data collection rates together with faster analysis would give more specific and quantitative information regarding the speed and strength of cellular responses to potential drug candidates.
Typical fluorescence imaging microplate readers use lamps and continuous wave argon-ion laser as excitation sources. In laser excitation systems, the intense collimated energy of the beam is expanded via a concave mirror and piano-convex cylindrical lens and rastered across the microplate hundreds of times per second with a faceted rotating drum scanner. Prior art fluorescence imaging instruments that use a laser excitation source are disclosed, for example in U.S. Pat. No. 5,355,215, in published U.S. Patent Application Publication No. US 2003/0223910 A1, in U.S. Patent Application Publication No. US 2002/0109100 A1, and in U.S. patent application Ser. No. 10/738,438, the entire contents of which are incorporated herein by this reference.
An advantage of the laser system is high energy output and very narrowband excitation. Disadvantages of the laser systems include (1) its large size (over 5′ long), (2) great weight; (3) its complexity (required several hours set-up and alignment by a skilled technician); (4) its cost (˜$25,000); (5) significant ongoing service requirements and infrastructure requirements (cooling water and 208V); and (5) limited wavelength range (typically 488 nm and 514 nm for an argon ion laser).